The osmolality of the cell suspension regulates phycobilisome-to-photosystem I excitation transfers in cyanobacteria.

The chlorophyll a (Chla) fluorescence of cyanobacteria, which at physiological temperature originates from photosystem (PS) II holochromes, is suppressed in hyperosmotic suspension, and enhanced in hypo-osmotic suspension (G.C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1-4). We investigated the mechanism of this phenomenon by comparing Synechococcus sp. PCC 7942 cells that had been treated with N-ethylmaleimide (NEM) in order to inhibit electronic excitation transfers from phycobilisomes (PBS) to Chlas of PSI (A.N. Glazer, Y.M. Gindt, C.F. Chan, K. Sauer, Photosynth. Res. 40 (1994) 167-173) with untreated control cells. The NEM-treated cells were indistinguishable from the control cells with regard to PSII-dependent oxygen evolution, reduction of post-PSII oxidants, and osmotically induced volume changes, but differed in the following properties: (i) they could not photoreduce post-PSI electron acceptors; (ii) they diverted more PBS excitation to PSII; (iii) the rise of Chla fluorescence upon light acclimation of darkened (state 2) cells was smaller; and (iv) the Chla fluorescence of light-acclimated (state 1) cells was insensitive to the cell suspension osmolality. These properties suggest that osmolality regulates the core-mediated excitation coupling between PBS and PSI, possibly by influencing mutual orientation and/or distance between core holochromes (ApcE, ApcD) and PSI holochromes. Thus, in hyper-osmotic suspension, PBS deliver more excitation to PSI (hence less to PSII); in hypo-osmotic cell suspension they deliver less excitation to PSI (hence more to PSII).     

Genetic background modifies amyloidosis in a mouse model of ATTR neuropathy

Penetrance and age of onset of ATTRV30M amyloidotic neuropathy varies significantly among different populations. This variability has been attributed to both genetic and environmental modifiers. We studied the effect of genetic background on phenotype in two lines of transgenic mice bearing the same ATTRV30M transgene. Amyloid deposition, transthyretin (TTR), megalin, clusterin and disease markers of endoplasmic reticulum stress, the ubiquitin-proteasome system, apoptosis, and complement activation were assessed with WB and immunohistochemistry in donor and recipient tissue. Our results indicate that genetic background modulates amyloid deposition by influencing TTR handling in recipient tissue and may partly account for the marked variability in penetrance observed in various world populations.     

A reappraisal of protein turnover values in neonates fed human milk or formula

We investigated the effect of human milk feeding on the nitrogen metabolism of appropriate-for-gestational age infants of birth weight 1.5-2.0 kg. Eight infants received pooled mature human milk. The remaining 20 were divided into two equal groups, who received one of two low-protein, milk-based formulae. The formulae were identical in composition except for the protein source, which was either casein- or whey-predominant. The three diet groups received similar total nitrogen (390 mg N.kg-1.d-1) and energy (500 kJ.kg-1.d-1) intakes. The human-milk-fed group, however, received a significantly higher intake of nonprotein and urea nitrogen and a significantly lower true protein nitrogen. Nitrogen metabolism was studied using a modified constant infusion of [15N]glycine, mixed with the feeding every 2-3 h. Urine was collected in approximately 3-h aliquots and analysed for total ammonia and urea nitrogen. Excretion of the 15N label was measured in urinary urea and ammonia. No differences were seen between the three diet groups in total [15N]urea or [15N]ammonia urinary excretion. However, the concentration of 15N in urinary urea in the human-milk-fed group was lower than in the two formula-fed groups. This reduction in concentration appeared due to a higher dietary intake of urea among the human-milk-fed group, and the consequent dilution of the label in the urine. As a result, protein turnover rates calculated from the [15N]urea end product were artificially raised in the milk-fed group, and were significantly higher than those in the formula groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

Cellular and tissue distribution of a single-strand-specific nuclease.

1. Specific antibodies which were raised against a single-strand-specific nuclease isolated from rat liver microsomes were used for characterizing this enzyme and determining its cellular and tissue distribution. 2. The single-strand-specific nuclease does not show any homology with other known nucleolytic enzymes. 3. It is mainly localized in microsomes and cytosol; traces of it are also found in nuclei, but it could not be detected in mitochondria. 4. Using the same specific antibodies we attempted to detect this nuclease in some other tissues which exhibit high metabolic rates, namely kidneys, heart and spleen. 5. Thus, with the aid of immunological techniques we were able to determine that at least part of the total poly(U) nucleolytic activity observed in kidney and heart is due to a nuclease immunologically identical to the enzyme under study. Kidneys were the best source for this enzyme.     

Guanidine-induced dissociation of mitochondrial F1-ATPase

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, and the fluorescence emission intensity of mitochondrial F1-ATPase was examined. Purified F1 from bovine heart mitochondria was slowly inactivated at low denaturant concentration, and inactivation was associated with delta and epsilon subunit dissociation. delta and epsilon subunits were bound together to form a stable and soluble heterodimer. In parallel, appearance of turbidity was observed. This was caused by the formation of alpha3beta3gamma non-covalent aggregates, as analyzed by SDS-PAGE. Short periods of exposition of the F1 complex to high concentrations of guanidine hydrochloride (0.8-3 M) again induced deltaepsilon dissociation as a heterodimer and the formation of an inactive alpha3beta3gamma subcomplex. This eventually dissociated progressively into single subunits caused by partial unfolding, as evidenced through changes of the protein intrinsic fluorescence emission. Our results suggest that the delta and epsilon subunits are loosely bound to alpha3beta3gamma , and play an important role in determining structural stability to isolated mitochondrial F1-ATPase.     

The Pseudomonas aeruginosa Isohexenyl Glutaconyl Coenzyme A Hydratase (AtuE) Is Upregulated in Citronellate-Grown Cells and Belongs to the Crotonase Family

Pseudomonas aeruginosa is one of only a few Pseudomonas species that are able to use acyclic monoterpenoids, such as citronellol and citronellate, as carbon and energy sources. This is achieved by the acyclic terpene utilization pathway (Atu), which includes at least six enzymes (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG) and is coupled to a functional leucine-isovalerate utilization (Liu) pathway. Here, quantitative proteome analysis was performed to elucidate the terpene metabolism of P. aeruginosa. The proteomics survey identified 187 proteins, including AtuA to AtuG and LiuA to LiuE, which were increased in abundance in the presence of citronellate. In particular, two hydratases, AtuE and the PA4330 gene product, out of more than a dozen predicted in the P. aeruginosa proteome showed an increased abundance in the presence of citronellate. AtuE (isohexenyl-glutaconyl coenzyme A [CoA] hydratase; EC 4.2.1.57) most likely catalyzes the hydration of the unsaturated distal double bond in the isohexenyl-glutaconyl-CoA thioester to yield 3-hydroxy-3-isohexenyl-glutaryl-CoA. Determination of the crystal structure of AtuE at a 2.13-Å resolution revealed a fold similar to that found in the hydratase (crotonase) superfamily and provided insights into the nature of the active site. The AtuE active-site architecture showed a significantly broader cavity than other crotonase superfamily members, in agreement with the need to accommodate the branched isoprenoid unit of terpenes. Glu139 was identified to be a potential catalytic residue, while the backbone NH groups of Gly116 and Gly68 likely form an oxyanion hole. The present work deepens the understanding of terpene metabolism in Pseudomonas and may serve as a basis to develop new strategies for the biotechnological production of terpenoids.     

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Biochemical analysis and structure determination of Paucimonas lemoignei poly(3‐hydroxybutyrate) (PHB) depolymerase PhaZ7 muteins reveal the PHB binding site and details of substrate–enzyme interactions

Five amino acids (Y105, Y176, Y189, Y189, W207) that constitute the substrate binding site of PHB depolymerase PhaZ7 were identified. All residues are located at a single surface‐exposed location of PhaZ7. Exchange of these amino acids by less hydrophobic, hydrophilic or negatively charged residues reduced binding of PhaZ7 to PHB. Modifications of other residues at the PhaZ7 surface (F9, Y66, Y103, Y124, Y169, Y172, Y173, F198, Y203, Y204, F251, W252) had no effect on substrate binding. The PhaZ7 wild‐type protein, three muteins with single amino acid exchanges (Y105A, Y105E, Y190E), a PhaZ7 variant with deletion of residues 202–208, and PhaZ7 in which the active‐site serine had been replaced by alanine (S136A) were crystallized and their structures were determined at 1.6–2.0 Å resolution. The structures were almost identical but revealed flexibility of some regions. Structural analysis of PhaZ7 (S136A) with bound 3‐hydroxybutyrate tetramer showed that the substrate binds in a cleft that is composed of Y105, Y176, Y189 and Y190 and thus confirmed the data obtained by site‐directed mutagenesis. To the best of our knowledge this is the first example in which the substrate binding site of a PHB depolymerase is documented at a molecular and structural level.